CN108196061B - Double-sandwich ELISA kit for detecting human PGRN based on monoclonal antibody - Google Patents

Abstract

The invention discloses a double-sandwich ELISA kit for detecting human PGRN based on monoclonal antibody, which comprises the following components: the kit comprises a 96-well ELISA plate, a coating antibody 4F10, a human PGRN standard protein, a biotin-labeled detection antibody 5F7-biotin, cross-linked protein of Avidin and HRP, a coating buffer solution, a confining solution, a sample and detection antibody diluent, an ELISA plate washing solution, a substrate solution and a stop solution. The PGRN protein content in the serum of cancer and diabetes patients can be rapidly detected, the multifunctional enzyme-linked immunosorbent assay device is used for carrying out quantitative analysis, the result is accurate and reliable, and a new auxiliary tool is provided for clinical examination and basic research.

Description

A double-sandwich ELISA kit for detecting human PGRN based on monoclonal antibody

technical field

The invention relates to a kit in the fields of biotechnology and medical detection research, in particular to a double-sandwich ELISA kit for detecting human PGRN based on a monoclonal antibody.

Background technique

The double-sandwich ELISA detection method is simple, efficient, reliable, and suitable for rapid screening of a large number of samples. The double-sandwich ELISA based on monoclonal antibodies has more clonal antibodies, which are more stable and specific.

Progranulin (PGRN) is a secreted glycoprotein containing 593 amino acids, containing 7.5 cysteine domains, named GrnP, GrnG, GrnF, GrnB, GrnA, GrnC, GrnD, GrnE. PGRN is closely related to physiological and pathological processes such as embryonic development, injury repair, inflammation, and tumor development. Studies have shown that the content of PGRN in human serum is related to some diseases, such as: diabetes, tumors, neurodegenerative diseases, etc. As a secreted glycoprotein, PGRN can be used as a marker for the diagnosis of these related diseases. , treatment, monitoring and prognosis provide an important basis, so it is particularly important to detect the content of PGRN protein in the serum of related patients quickly, accurately and with high sensitivity.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a double-sandwich ELISA kit for detecting human PGRN based on monoclonal antibody, which is simple, has high sensitivity, strong specificity and is suitable for large-scale detection.

In order to realize the above-mentioned tasks, the present invention is realized through the following technical solutions:

A double-sandwich ELISA kit for detecting human PGRN based on monoclonal antibodies, characterized in that the contents of the kit are composed as follows: 96-hole ELISA plate, coating antibody 4F10, human PGRN standard protein, biotin-labeled Detection antibody 5B7-biotin, cross-linked protein of Avidin and HRP, coating buffer, blocking solution, sample and detection antibody dilution, ELISA plate washing solution, substrate solution and stop solution.

According to the present invention, the coating antibody 4F10 is a mouse anti-human monoclonal antibody directed against the GrnA domain in human PGRN, and the working concentration is 10 μg/mL.

Further, the biotin-labeled detection antibody 5B77-biotin is a mouse anti-human monoclonal antibody against the GrnB domain in human PGRN, and the working dilution ratio is 1:4000.

Described coating buffer is 0.05M carbonate buffer, pH is 9.6;

The blocking solution is a phosphate buffer containing 10% calf serum in volume fraction, and the pH is 7.4;

The sample and detection antibody diluent are phosphate buffered saline containing 1% calf serum, and the pH is 7.4;

The ELISA plate washing solution is a phosphate buffer containing a volume fraction of 0.1% Tween-20, and the pH is 7.4;

The substrate solution is TMB, namely tetramethylbenzidine (3,3',5,5'-Tetramethylbenzidine) substrate buffer;

The stop solution is a concentrated sulfuric acid solution with a molar concentration of 2M.

The human PGRN standard protein is obtained by the method of eukaryotic protein purification; the cross-linked protein of Avidin and HRP is the cross-linking of purified Avidin and HRP protein by the sodium periodate method.

The described sodium periodate method is to react HRP with sodium periodate (NaIO ) to obtain activated HRP;

The Avidin reacts with activated HRP to obtain a cross-linked protein of Avidin and HRP.

According to the test of the applicant, the double-sandwich ELISA kit for detecting human PGRN based on the monoclonal antibody of the present invention can be used for the serological detection application of breast cancer, lung cancer and type II diabetes patients.

The serological detection of the breast cancer, lung cancer and type II diabetes patients is to detect the recombinant PGRN protein and the natural human PGRN protein.

The detection of recombinant PGRN protein and natural human PGRN protein specifically includes the following steps:

(1) Coating ELISA plate: Dilute the coating antibody 4F10 with coating buffer to 10 μg/mL, 100 μL/well, overnight at 4°C;

(2) Blocking: shake off the liquid in the well, add 200 μL/well of ELISA plate washing solution, wash 3-5 times, add 200 μL/well of blocking solution, wash 3-5 times at 37°C for 2 hours, and spin dry;

(3) Add the sample to be detected: add the sample to be detected to the ELISA plate, 100 μL/well, wash 3-5 times at 37°C for 1 hour, and spin dry;

(4) Add detection antibody: add 5B7-biotin to the ELISA plate, 100 μL/well, wash 3-5 times at 37°C for 1 hour, and spin dry;

(5) Add the cross-linked protein of Avidin and HRP: add the cross-linked protein of Avidin and HRP to the microtiter plate, 100 μL/well, wash 5-6 times at 37°C for 1 hour, and spin dry;

(6) Color development: Add substrate solution to the ELISA plate, 100 μL/well, 37° C., 3-5 min.

(7) Stop: Add stop solution to the microplate plate, 50 μL/well, after the reaction, read at 450nm of the microplate reader.

Compared with the prior art, the double-sandwich ELISA kit for detecting human PGRN based on monoclonal antibody of the present invention has the following beneficial technical effects:

(1) The coated antibody and enzyme-labeled antibody used are both monoclonal antibodies against PGRN, and more cloned antibodies are more stable and specific;

(2) The content of PGRN protein in the serum of cancer and diabetes patients can be quickly detected, and the quantitative analysis is carried out by a multi-function microplate reader. The results are accurate and reliable, and it provides a new auxiliary tool for clinical examination and basic research.

Description of drawings

Fig. 1 is the antibody optimal paired screening suitable for detecting human PGRN double-sandwich ELISA kit;

Fig. 2 is the optimization of the double-sandwich ELISA kit based on monoclonal antibody detection of human PGRN;

Fig. 3 is the determination of the detection sensitivity of the double-sandwich ELISA kit for detecting human PGRN based on monoclonal antibody;

Figure 4 shows the results of double-sandwich ELISA kits for detecting PGRN protein in serum of different populations, in which, in panel A, NGT is a non-diabetic population, T2D is a type II diabetic patient, in panel B, Breast cancer group is a breast cancer population, lungcancer is a Lung cancer population, Health group as healthy controls.

The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

Detailed ways

This example provides a double-sandwich ELISA kit for detecting human PGRN based on monoclonal antibodies.

First, the use of monoclonal antibodies is to use hybridoma cell technology to immunize mice with Grns proteins in each domain of human PGRN, fuse the spleen cells of the immunized mice with SP2/0 myeloma cells, and undergo cell cloning and indirect ELISA. The obtained 11 mouse anti-human monoclonal antibodies against each domain of human PGRN: 4G3, 2E2, 1G6, 4E8, 5B7, 4F10, 2A5, 3C5, 3F9, 1D5, 3F7; Biotin labeling, the coating antibody and enzyme-labeled antibody were paired with double sandwich antibody respectively, and the best monoclonal antibody pairing was screened: 4F10 (for GrnA domain) and 5B7 (for GrnB domain)

In the second aspect, the double-sandwich ELISA kit for detecting human PGRN based on monoclonal antibodies provided in this example, the contents of the kit are composed as follows: 96-well ELISA plate, coating antibody 4F10, human PGRN standard protein, Biotin-labeled detection antibody 5B7-biotin, cross-linked protein of Avidin and HRP, coating buffer, blocking solution, sample and detection antibody diluent, ELISA plate washing solution, substrate solution and stop solution.

in:

(1) The optimal working concentration of the coated antibody 4F10 is 10 μg/mL;

(2) human PGRN standard protein, obtained by the method of eukaryotic protein purification;

(3) Biotin-labeled detection antibody 5B7-biotin, obtained by a biotin-labeling kit (sold by Thermo Company), the optimal dilution ratio for work: 1:4000.

(4) The cross-linked protein of Avidin and HRP, through the sodium periodate method, HRP reacts with sodium periodate (NaIO4) to obtain activated HRP; Avidin reacts with activated HRP to obtain the cross-linked protein of Avidin and HRP.

(5) Coating buffer, 0.05M carbonate buffer (pH 9.6);

(6) Blocking solution, phosphate buffered saline (pH 7.4) containing 10% calf serum by volume;

(7) Sample and detection antibody diluent, phosphate buffered saline (pH 7.4) containing 1% calf serum by volume;

(8) ELISA plate washing solution, containing PBS (PBST, pH 7.4) with a volume fraction of 0.1% Tween-20;

(9) substrate solution, substrate buffer containing TMB;

In the third aspect, the double-sandwich ELISA kit for detecting human PGRN based on monoclonal antibody provided in this example can be used for serological detection of breast cancer, lung cancer and type II diabetes patients. That is, the recombinant PGRN protein and the natural human PGRN protein are detected.

The detection of recombinant PGRN protein and natural human PGRN protein specifically includes the following steps:

(1) Coating ELISA plate: Dilute the coating antibody 4F10 with coating buffer, 100 μL/well, overnight at 4°C;

(2) Blocking: shake off the liquid in the well, add 200 μL/well of ELISA plate washing solution, wash 3-5 times, add 200 μL/well of blocking solution, wash 3-5 times at 37°C for 2 hours, and spin dry;

(3) Add the sample to be detected: add the sample to be detected to the ELISA plate, 100 μL/well, wash 3-5 times at 37°C for 1 hour, and spin dry;

(4) Add detection antibody: add 5B7-biotin to the ELISA plate, 100 μL/well, wash 3-5 times at 37°C for 1 hour, and spin dry;

(5) Add the cross-linked protein of Avidin and HRP: add the cross-linked protein of Avidin and HRP to the microtiter plate, 100 μL/well, wash 5-6 times at 37°C for 1 hour, and spin dry;

(6) Color development: Add substrate solution to the ELISA plate, 100 μL/well, 37° C., 3-5 min.

(7) Stop: Add stop solution to the microplate plate, 50 μL/well, after the reaction, read at 450nm of the microplate reader.

The following are specific examples given by the inventor, and the present invention is not limited to the following examples.

Example 1: Screening of double sandwich ELISA antibody pairings

(1) Coating antibody: The purified monoclonal antibodies against each domain of human PGRN anti-GrnG (4G3, 2E2), anti-GrnF ((1G6, 4E8), anti-GrnB (5B7), anti-GrnA (4F10), anti-GrnC (2A5), anti-GrnD (3C5, 3F9), anti-GrnE (1D5, 3F7), diluted to 10 μg/mL, coated with 100 μL/well, overnight at 4°C;

(2) Blocking: shake off the liquid in the well, add 200 μL/well of ELISA plate washing solution, wash 3-5 times, add 200 μL/well blocking solution, wash 3-5 times at 37°C for 2 hours, and spin dry;

(3) Add PGRN protein sample: add PGRN protein sample (0.5 μg/mL) to the ELISA plate, 100 μL/well, wash 3-5 times at 37° C. for 1 h, and spin dry;

(4) Add detection antibody: Add biotin-labeled monoclonal antibody to the ELISA plate, 100 μL/well, wash 3-5 times at 37°C for 1 hour, and spin dry;

(5) Add the cross-linked protein of Avidin and HRP: add the cross-linked protein of Avidin and HRP to the microtiter plate, 100 μL/well, wash 5-6 times at 37°C for 1 hour, and spin dry;

(6) Color development: add substrate solution to the ELISA plate, 100 μL/well, 37°C, 3-5min;

(7) Stop: Add stop solution to the microplate plate, 50 μL/well, after the reaction, read at 450nm of the microplate reader.

The pairing results are shown in Figure 1, wherein the optimal paired antibodies for the double-sandwich ELISA kit for detecting human PGRN are: the coated antibody 4F10 and the biotin-labeled detection antibody 5B7-biotin.

Example 2: Cross-linking of Avidin to HRP protein

Avidin was cross-linked with HRP by the sodium periodate method, specifically:

First, HRP reacts with sodium periodate (NaIO4) to obtain activated HRP; Avidin reacts with activated HRP to obtain a cross-linked protein of Avidin and HRP.

Example 3: Optimization of Double Sandwich ELISA Kit

(1) Optimization of coating antibodies

The monoclonal antibody 4F10 against GrnA was used as the coating antibody, and the monoclonal antibody 4F10 was coated with a concentration gradient of 40 μg/mL, 20 μg/mL, 10 μg/mL, 5 μg/mL, and 2.5 μg/mL. After blocking, PGRN protein was added. 0.1μg/mL reaction for 1h, add the detection antibody 5B7-Biotin to the detection well, wash 3-5 times after 1h at 37℃, add Avidin-HRP to the reaction well, wash 5-6 times after 37℃, 1h , add the substrate solution for color development, add stop solution to the reaction well after 3-5min reaction, and measure the OD value at 450nm. Measured OD value minus blank OD value is the best coating antibody concentration, the results are shown in Figure A of Figure 2, 10 μg/mL is the best coating concentration.

(2) Optimization of detection antibodies

The detection antibody is the monoclonal antibody 5B7 against GrnB, and the 5B7 antibody is labeled with biotin to obtain 5B7-Biotin. The concentration of the detection antibody Biotin-5B7 was optimized, and Biotin-5B7 was serially diluted according to 1/250, 1/1000, 1/2000, 1/4000, 1/8000, 1/16000, 1/32000, according to the above method. test. The optimal dilution concentration is selected to measure the maximum OD value and the lowest concentration of the detection antibody. The results are shown in Figure B of Figure 2, and 1/4000 is the optimal dilution concentration.

(3) Determination of detection sensitivity

After coating and blocking with the optimized coating antibody concentration, add the PGRN eukaryotic expression protein diluted in gradient, wash 3-5 times at 37°C for 1 h, and detect the antibody (1:4000) in 5B7-Biotin in the well to be tested. , after 37℃, 1h, wash 3-5 times, add Avidin-HRP cross-linked protein respectively, after 37℃, 1h, wash 5-6 times, then add substrate solution for reaction, measure at 450nm, the results are shown in the figure 3, the detectable sensitivity is 125pg/mL-63pg/mL, and the detectable range is 8ng/mL-125pg/mL.

Embodiment 5: Detection method of double sandwich ELISA kit based on monoclonal antibody detection of human PGRN

(1) Coating ELISA plate: Dilute the coating antibody 4F10 to 10 μg/mL in coating buffer, 100 μL/well, overnight at 4°C;

(2) Blocking: shake off the liquid in the well, add 200 μL/well of ELISA plate washing solution, wash 3-5 times, add 200 μL/well of blocking solution, wash 3-5 times at 37°C for 2 hours, and spin dry;

(3) Add the sample to be tested: Dilute the sample to be tested appropriately, add it to the ELISA plate, 100 μL/well, 37°C for 1 h, wash 3-5 times, and spin dry;

(4) Add detection antibody: add 5B7-biotin to the ELISA plate, 100 μL/well, wash 3-5 times at 37°C for 1-2 hours, and spin dry;

(5) Add the cross-linked protein of Avidin and HRP: add the cross-linked protein of Avidin and HRP to the microtiter plate, 100 μL/well, wash 5-6 times at 37°C for 1 hour, and spin dry;

(6)) Color development: add substrate solution to the ELISA plate, 100 μL/well, 37°C, 3-5min;

(7) Termination: Add stop solution to the microtiter plate, 50 μL/well, after the reaction, read at 450nm of the microplate reader.

Example 6: Application of double-sandwich ELISA kit for detection of human PGRN based on monoclonal antibody

The ELISA method established in Example 5 was used to detect 30 cases of cancer patients (samples from Fenyang Hospital in Shanxi Province, including 23 cases of lung cancer and 7 cases of breast cancer) and 30 cases of normal healthy people (healthy people were selected from the physical examination samples of students from Shaanxi Normal University) ); 205 patients with type Ⅱ diabetes (samples were from Nanjing First People’s Hospital) and 78 normal healthy people (healthy people were selected from the physical examination samples of students from Shaanxi Normal University), PGRN recombinant protein was used as a positive control, according to the obtained standard OD A standard curve was drawn between the values and the corresponding concentrations, and the serological levels of PGRN protein in each population were calculated.

Results As shown in Figure 4, the serological levels of PGRN protein in the breast cancer, lung cancer and diabetes groups were significantly higher than those in the normal people, and the difference was statistically significant (p<0.001).

Claims (2)

1. a double-sandwich ELISA kit based on monoclonal antibody detection human PGRN, is characterized in that, the content of this test kit is formed as follows: 96 holes ELISA plate, coating antibody 4F10, human PGRN standard protein, biotin Labeled detection antibody 5B7-biotin, cross-linked protein of Avidin and HRP, coating buffer, blocking solution, sample and detection antibody dilution, ELISA plate washing solution, substrate solution and stop solution; The coating antibody 4F10 is a mouse anti-human monoclonal antibody against the GrnA domain in human PGRN, and the working concentration is 10 μg/mL; The biotin-labeled detection antibody 5B7-biotin is a mouse anti-human monoclonal antibody against the GrnB domain in human PGRN, and the working dilution ratio is 1:4000; Described coating buffer is 0.05M carbonate buffer, pH is 9.6; The blocking solution is a phosphate buffer containing 10% calf serum in volume fraction, and the pH is 7.4; The sample and detection antibody diluent are phosphate buffered saline containing 1% calf serum, and the pH is 7.4; The ELISA plate washing solution is a phosphate buffer containing a volume fraction of 0.1% Tween-20, and the pH is 7.4; The substrate solution is TMB; Described stop solution is the vitriol oil solution that molar concentration is 2M; The human PGRN standard protein is obtained by the method of eukaryotic protein purification; the cross-linked protein of Avidin and HRP is the cross-linking of purified Avidin and HRP protein by the sodium periodate method; The described sodium periodate method is to react HRP with sodium periodate (NaIO ) to obtain activated HRP; The Avidin reacts with activated HRP to obtain a cross-linked protein of Avidin and HRP. 2. the application of the double-sandwich ELISA kit based on monoclonal antibody detection of human PGRN according to claim 1 for the preparation of breast cancer, lung cancer and type Ⅱ diabetes serological detection kit; described breast cancer, lung cancer and Ⅱ Serological detection of type diabetes patients is to detect recombinant PGRN protein and natural human PGRN protein; The detection of recombinant PGRN protein and natural human PGRN protein specifically includes the following steps: (1) Coating ELISA plate: Dilute the coating antibody 4F10 with coating buffer to 10 μg/mL, 100 μL/well, overnight at 4°C; (2) Blocking: shake off the liquid in the well, add 200 μL/well of ELISA plate washing solution, wash 3-5 times, add 200 μL/well of blocking solution, wash 3-5 times at 37°C for 2 hours, and spin dry; (3) Add the sample to be detected: add the sample to be detected to the ELISA plate, 100 μL/well, wash 3-5 times at 37°C for 1 hour, and spin dry; (4) Add detection antibody: add 5B7-biotin to the ELISA plate, 100 μL/well, wash 3-5 times at 37°C for 1 hour, and spin dry; (5) Add the cross-linked protein of Avidin and HRP: add the cross-linked protein of Avidin and HRP to the microtiter plate, 100 μL/well, wash 5-6 times at 37°C for 1 hour, and spin dry; (6) Color development: Add substrate solution to the ELISA plate, 100 μL/well, and add stop solution after 3-5 min at 37°C; (7) Termination: Add stop solution to the microtiter plate, 50 μL/well, after the reaction, read at 450nm of the microplate reader.

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